Comparative analysis in Papaver species- specialization of biosynthetic enzymes exemplified by the evolution of substrate specifity of O-methyltransferases

WOLFGANG BRANDT
LUDGER A. WESSJOHANN
Leibniz Institute of Plant Biochemistry
Weinberg 3
D-06120 Halle/S.
Wolfgang.Brandt@ipb-halle.de
Ludger.Wessjohann@ipb-halle.de
http://www.ipb-halle.de/de/forschung/natur-und-wirkstoffchemie/

The benzylisoquinoline alkaloids such as the analgesic morphine, the antimicrobial berberine, the vasodilator papaverine and the antitussivum codeine are the major secondary metabolites of the plant order of the Ranunculales, and especially of the plant family of the Papaveraceae. There are about 2,500 known benzylisoquinoline structures, which are all derived biosynthetically from a central core structure. This structural complexity is achieved by a multitude of specific oxidations, reductions / dehydrogenations and methylations. Three cDNAs coding for O-methyltransferases (OMTs) have previously been isolated and characterized from Papaver somniferum with respect to substrate specificity. The presence of enzymes methylating benzylisoquinolines as well as phenolic substances among those specific for benzylisoquinolines led to the assumption that the benzylisoquinoline specific OMTs are evolutionary derived from methyltransferases of phenylpropanoid metabolism which adopted their present specificity by modification of the substrate binding site. The objective of the project is the identification of critical amino acids conferring the substrate specificity of benzylisoquinoline methylating OMT from P. somniferum. A new OMT with high homology to benzylisoquinoline OMT was recently isolated based on its specific expression in P. somniferum compared to other Papaver species. A model of the tertiary structure model of the unknown P. somniferum OMT was created followed by substrate docking experiments in order to obtain hints about its substrate specificity. Assays performed with the heterologuously expressed enzyme confirmed the results of the docking studies. The project involves the extension of the modelling approach to other benzylisoquinoline OMTs as a basis for site directed mutagenesis studies to evaluate the evolutionary relationship.

References:
Ziegler, J., Voigtländer, S., Schmidt, J., Kramell, R., Miersch, O., Ammer, C., Gesell, A., Kutchan, T.M. (2006) Comparative transcript and alkaloid profiling in Papaver species identifies a short chain dehydrogenase/reductase involved in morphine biosynthesis. Plant J. 48: 177-192.

Ziegler, J., Diaz-Chavez, M.L., Kramell, R., Ammer, C., Kutchan, T.M. (2005) Comparative macroarray analysis of morphine containing Papaver somniferum and eight morphine free Papaver species identifies an O-methyltransferase involved in benzylisoquinoline biosynthesis. Planta 222(3):458-471.

Geissler, R.; Brandt, W.; Ziegler, Molecular modeling and site-directed mutagenesis reveal the benzylisoquinoline binding site of the short-chain dehydrogenase/reductase salutaridine reductase. J. Plant Physio,l 2007, 143, 1493-1503.

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